PAL is a non-mammalian enzyme widely distributed in plants (Koukol, et al., J. Biol. Chem. 236:2692-2698 (1961); Hanson, et al., The Enzymes 7:75-166 (1972); Poppe, et al., Curr. Org. Chem. 7:1297-1315 (2003)), some fungi (Rao, et al., Can. J. Biochem. 4512:1863-1872 (1967); Abell, et al., Methods Enzymol. 142:242-253 (1987)) and bacteria (Bezanson, et al., Can. J. Microbiol. 16:147-151 (1970); Xiang, et al., J. Biol. Chem. 277:32505-32509 (2002); Hill, et al., Chem. Commun. 1358-1359 (2003)) and can be recombinantly produced in Escherichia coli. 
A representative list of PALs includes: Q9ATN7 Agastache rugosa; O93967 Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4 Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Giant timber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726 Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle) (Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5, Q9XFX6 Citrus clementina x Citrus reticulate; Q42667 Citrus limon (Lemon); Q8H6V9, Q8H6W0 Coffea canephora (Robusta coffee); Q852S1 Daucus carota (Carrot); O23924 Digitalis lanata (Foxglove); O23865 Daucus carota (Carrot); P27991 Glycine max (Soybean); O04058 Helianthus annuus (Common sunflower); P14166, Q42858 Ipomoea batatas (Sweet potato); Q8GZR8, Q8W2E4 Lactuca sativa (Garden lettuce); O49835, O49836 Lithospermum erythrorhizon; P35511, P26600 Lycopersicon esculentum (Tomato); P35512 Malus domestica (Apple) (Malus sylvestris); Q94C45, Q94F89 Manihot esculenta (Cassava) (Manioc); P27990 Medicago sativa (Alfalfa); P25872, P35513, P45733 Nicotiana tabacum (Common tobacco); Q6T1C9 Quercus suber (Cork oak); P14717, P53443, Q7M1Q5, Q84VE0, Q84VE0 Oryza sativa (Rice); P45727 Persea americana (Avocado); Q9AXI5 Pharbitis nil (Violet) (Japanese morning glory); P52777 Pinus taeda (Loblolly pine); Q01861, Q04593 Pisum sativum (Garden pea); P24481, P45728, P45729 Petroselinum crispum (Parsley) (Petroselinum hortense); Q84LI2 Phalaenopsis×Doritaenopsis hybrid cultivar; P07218, P19142, P19143 Phaseolus vulgaris (Kidney bean) (French bean); Q7XJC3, Q7XJC4 Pinus pinaster (Maritime pine); Q6UD65 Populus balsamifera subsp. trichocarpa×Populus deltoides; P45731, Q43052, 024266 Populus kitakamiensis (Aspen); Q8H6V5, Q8H6V6 Populus tremuloides (Quaking aspen); P45730 Populus trichocarpa (Western balsam poplar); O64963 Prunus avium (Cherry); Q94EN0 Rehmannia glutinosa; P11544 Rhodosporidium toruloides (Yeast) (Rhodotorula gracilis); P10248 Rhodotorula rubra (Yeast) (Rhodotorula mucilaginosa); Q9M568, Q9M567 Rubus idaeus (Raspberry); P31425, P31426 Solanum tuberosum (Potato); Q6SPE8 Stellaria longipes (Longstalk starwort); P45732 Stylosanthes humilis (Townsville stylo); P45734 Trifolium subterraneum (Subterranean clover); Q43210, Q43664 Triticum aestivum (Wheat); Q96V77 Ustilago maydis (Smut fungus); P45735 Vitis vinifera (Grape); and Q8VXG7 Zea mays (Maize).
Numerous studies have focused on the use of the enzyme phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) for enzyme substitution treatment of phenylketonuria (PKU) (Hoskins, et al., Lancet 1(8165):392-394 (1980); Gilbert, et al., Biochem. J. 199(3):715-723 (1981); Hoskins, J. A., et al., Res. Commun. Chem. Pathol. Pharmacol. 35(2):275-282 (1982); Sarkissian, et al., Proc. Natl. Acad. Sci. USA 96(5):2339-2344 (1999); Liu, et al., Artif. Cells Blood Substit. Immobil. Biotechnol. 30(4):243-257 (2002); Wieder, et al., J Biol. Chem. 254(24):12579-12587 (1979); Gamez, et al., Mol. Ther. 11(6):986-989 (2005); Ambrus, et al., J. Pharmacol. Exp. Ther. 224(3):598-602 (1983); Ambrus, et al., Science 201(4358):837-839 (1978); Kalghatgi, Res. Commun. Chem. Pathol. Pharmacol. 27(3):551-561 (1980); Ambrus, Res. Commun. Chem. Pathol. Pharmacol. 37(1):105-111 (1982); Gilbert, et al., Biochem. Biophys. Res. Commun. 131(2):557-563 (1985); Pedersen, Res. Commun. Chem. Pathol. Pharmacol. 20(3):559-569 (1978); Marconi, et al, Biochimie 62(8-9):575-580 (1980); Larue, et al., Dev. Pharmacol. Ther. 9(2):73-81 (1986); Ambrus, et al., Ann. Intern. Med. 106(4):531-537 (1987); Bourget, et al., Appl. Biochem. Biotechnol. 10:57-59 (1984); Bourget, et al., FEBS Lett. 180(1):5-8 (1985); Bourget, et al., Biochim. Biophys. Acta 883(3):432-438 (1986); Chang, et al., Artif. Cells Blood Substit. Immobil. Biotechnol. 23(1):1-21 (1995); Chang, et al., Mol. Biotechnol. 17(3):249-260 (2001); U.S. Pat. No. 5,753,487).
The use of PAL for cancer treatment has been suggested based on its ability to limit the nutrient supply of phenylalanine to cancer cells and thereby inhibit neoplastic growth (Fritz, et al., J. Biol. Chem. 251(15):4646-4650 (1976); Roberts, et al., Cancer Treat. Rep. 60(3):261-263 (1976); Shen, et al., Cancer Res. 37(4):1051-1056 (1977); Shen, et al., Cancer Treat. Rep. 63(6):1063-1068 (1979); Wieder, et al., J. Biol. Chem. 254(24):12579-12587 (1979)). In addition, PAL-mediated reduction in phenylalanine prevented the proliferation of murine leukemia and metastatic melanoma. However, intravenously injected pegylated PAL was cleared rapidly from circulating blood after the 13th injection (Abell, et al., Cancer Res. 33:2529-2532 (1973); Roberts, et al., (1976), ibid.; Shen, et al., (1977), ibid.; (Shen, et al., J. Reticuloendothelial Soc. 23:167-175 (1978)).
Certain neoplastic or cancer cells have a higher metabolic rate and a greater requirement than normal cells for essential amino acids such as phenylalanine. There is evidence in the literature suggesting that restriction or reduction of specific amino acids, e.g., phenylalanine, through the use of amino acid degrading enzymes, e.g., PAL, may reduce the growth of certain tumor cells in human cancer patients and in animal models. For example, certain leukemic cells lack the enzyme asparagine synthetase, which synthesizes the non-essential amino acid asparagine from glutamine, and are thus dependent upon asparagine for survival. Oncaspar (pegaspargase, Enzon Pharmaceuticals, Inc.), a pegylated L-asparaginase, has been used successfully to treat acute lymphoblastic leukemia (ALL) (Graham, Adv. Drug Del. Rev. 55:1293-1302 (2003)). Other examples of amino acids as potential targets for enzymatic depletion in cancer therapy include glutamine (glutamine deaminase, Medical Enzymes AG), arginine (arginine deiminase, Phoenix Pharmacologics, Inc.) and methionine (methioninase, Anticancer, Inc.) (See, for example, U.S. Pat. Nos. 6,312,939, 6,737,259 and 5,690,929).
Dietary restriction of phenylalanine has been shown to inhibit growth and metastasis of highly invasive metastatic melanoma and androgen independent prostate cancer cells in animal models, promote apoptosis of tumor, but not normal, cells in culture, increase survival of tumor-bearing mice, sensitize tumor cells to chemotherapeutic agents, and augment cytotoxicity by toxins (Fu, et al., Nutr. Cancer 31:1-7 (1998); Fu, et al., Cancer Res. 59:758-765 (1999); Fu, et al., Nutr. Cancer 45:60-73 (2003); Fu, et al., J. Cell. Physiol. 209:522-534 (2006); Meadows, et al., Cancer Res. 42:3056-3063 (1982); Elstad, et al., Anticancer Res. 13:523-528 (1993); Elstad, et al., Nutr. Cancer 25:47-60 (1996); Nunez, et al., Cancer Lett. 236:133-141 (2006)).
Enyzmatic depletion of phenylalanine using PAL from the yeast Rhodosporidium toruloides (also known as Rhodotorula glutinis) (RtPAL) inhibited the growth of leukemic lymphocytes in culture in vitro (Abell, et al., Cancer Res. 32:285-290 (1972); Stith, et al., Cancer Res. 33:966-971 (1973)) and in mice in vivo (Abell, et al., Cancer Res. 33:2529-2532 (1973)). However, after repeated injections into mice, the clearance of RtPAL from plasma was greatly accelerated, and the clearance rate was more rapid in tumor bearing, as compared to non-tumor bearing, mice (Fritz, et al., J. Biol. Chem. 251:4646-4650 (1976); Shen, et al., Cancer Res. 37:1051-1056 (1977)). The half-life of RtPAL was decreased to about 1 hour after multiple administration due to an increase in antibody titer, demonstrating that total body radiation may be necessary to delay clearance and enhance half-life (Shen, et al., J. Reticuloendothelial Soc. 23:167-175 (1978).
RtPAL has been pegylated in an attempt to reduce the enzyme's immunogenicity and clearance rate in vivo (Wieder, et al., J. Biol. Chem. 254:12579-12587 (1979)). After a single intravenous injection or after multiple intravenous injections into mice, the blood half-life of pegylated RtPAL was longer than unpegylated RtPAL; however, the pegylated RtPAL was still rapidly cleared from the blood after the thirteenth intravenous injection.
Although PAL potentially has various therapeutic applications, the use of PAL may be limited by reduced specific activity and proteolytic instability. Similar to other therapeutic proteins, use of PAL as an enzyme therapy is accompanied by several disadvantages such as immunogenicity and proteolytic sensitivity (see Vellard, Curr. Opin. Biotechnol. 14:1-7 (2003)). As yet, a concerted effort toward improving these parameters has not been made due to a paucity of structural and biochemical knowledge regarding this protein. Thus, there remains a need for PAL molecules with optimal kinetic characteristics, including potent catalytic activity, greater biological half-life, greater biochemical stability, and/or attenuated immunogenicity, for therapeutic use, including the treatment of cancer.